June 25, 2007

Preparation of Ultra-competent Cells Protocol -- Inoue Method

--Prepared competent cells of DB3.1 and DH5a
--When centrifuging, centrifuge used only held max 50ml, so spun down the 250ml culture in two separate centrifuge tubes with 40ml culture (don't fill all the way to the top to prevent spilling) for ~5minutes each time.

TB Solution
*10mM PIPES
*15mM CaCl2
*250mM KCl
*Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add
55mM MnCl2. Adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C

June 28, 2007

DH5a and DB3.1 Competent Cells (cont.)

--Transformed cells grew well
--Very few pUC19 cells (probably due to very small amount of vector added during transformation)
--No cells on negative control (good)
--Inoculated transformed cells into cell cultures for mini-prepping (pUC19 into 2mL, death gene into 15mL)

PCR

--PCR testing out Taq and Pfu DNA polymerase (during this experiment, thought Pfx was being used instead of Pfu) on the following primers (12 samples total))

Gel extraction

dye seems to stay and go through so that the liquid in the centrifuge tube is blue. Dye might possible interfere with restriction?? Also, a lot of end volume, so the product iws more dilute than through gel extraction

Today's gel
--Extracted both xyl promoter and xylR total using gel extraction kit